Impact of Single Strand Recombinases and Transposases on Genome Dynamics – MOBISING

The project involves a wet bench approach including genetic, biochemical, biophysical and structural studies to investigate their detailed molecular mechanisms and to understand their impact on genome dynamics. This will be strengthened by bioinformatics to explore their target sites, diversity and distribution. In particular, the phylogenetic analysis of REP-associated HuH enzyme and REP sequences will provide important information on their evolution.

We have obtained some encouraging results in the REP and ISCR studies. Our REP phylogenetic analysis is also in progress.

This project will provide an overview of the role of HuH-mediated recombination in genome plasticity and evolution together with a precise description of the accompanying mechanisms.

Michael Chandler1*, Fernando de la Cruz2, Fred Dyda3, Alison B. Hickman3, Gabriel Moncalian2, Bao Ton-Hoang1. Breaking and joining single-stranded DNA: the HUH endonuclease superfamily. Nature Reviews Microbiology, July 2013.

Transposable elements (TE) are important in genome evolution and central to the bacterial horizontal gene pool. They are extremely diverse and can constitute a large proportion of prokaryote genomes. Transposition requires a specific TE-encoded transposase (Tpase) for DNA cleavage and transfer. Tpases are the most abundant genes in the public sequence databases. Insertion sequences (IS) are the simplest autonomous TE. There are more than 3700 different complete ISs in the international IS register, ISfinder (www-IS.biotoul.fr), a number which is growing continuously and rapidly. Besides direct involvement in TE mobility, in eukaryotes, a number of Tpases have been domesticated to carry out diverse cellular functions. In prokaryotes, such domestication has not yet been extensively identified. This project addresses the central role of a functionally related group of transposable elements called “HuH elements” in structuring and shaping bacterial genomes. We have identified and characterised a widespread class of IS (IS200/IS605 family) quite different from classical TEs: they use obligatory single strand DNA intermediates and have ends with subterminal imperfect palindromes (IP) which are recognised and bound by their Tpases. These Tpases, with a catalytic site containing a single Tyr and a His-u-His amino acid triad, are members of a larger “HuH” endonuclease superfamily including RCR Rep proteins (Rolling Circle Replication), relaxases (conjugal plasmid transfer) and Tpases of the IS91 family. All create a covalent 5'-phosphotyrosine enzyme-substrate intermediate. IS200/IS605 family Tpases are small and form a dimer with two shared active sites: an HuH motif from one monomer and a catalytic Tyr from the other. The dimer binds the subterminal IP located some distance from the cleavage sites. Remarkably, cleavage sites are not recognised directly by the protein but by short “guide” sequences 5' to the IP foot. Recognition involves a network of canonical and non-canonical base interactions similar to those found in RNA structures. We have demonstrated the importance of the lagging strand template for activity of some members and our in silico genomic analysis suggests that all IS200/IS605 family members have evolved a mode of transposition that exploits ssDNA at the replication fork. Some members of this group appear to have been domesticated to perform important roles in the prokaryotic cell: such as homing endonucleases in some group I prokaryotic introns and as enzymes responsible for proliferation of short intergenic multicopy palindromic regulatory sequences called REPs. Other distinct but functionally related Tpases are encoded by ISCR elements, recently shown to be major players in sequestrating and transmitting multidrug resistance in bacteria. We will examine the role of these “HuH” enzymes and their impact on their bacterial host genomes. The project is divided into 4 closely related and complementary areas: 1) obtaining a detailed description of the mechanism of IS608 transposition including replication fork targeting and dynamics of the single strand transpososome, the nucleoprotein machine which assures transposition; 2) understanding the role of Tpase domestication in IStron mobility and in REP/BIME expansion in genome structure; 3) exploration of ISCR transposition to obtain insights into gene capture and 4) development and exploitation of the ISfinder bioinformatics platform, to generate a detailed high quality annotation of bacterial genomes for these particular types of IS-related elements. We expect the ensemble of these results to provide an overview of the role of “HuH” transposases and their associated mobile elements in shaping the prokaryotic genome. This should supply a firm and essential base on which to build further studies to understand how genome structuring by this IS family may be regulated and integrated dynamically into the growing cell.